We propose a novel approach to identify probe combinations to discriminate and purify primitive and immature murine clonogenic cells committed to myelopoiesis. Purified clonogenic cell preparations will facilitate numerous biologic and clinical studies such as gene manipulations on a single cell basis, cell kinetic- based modulation of drug-induced hemopoietic toxicity, and regulation of cell proliferation and differentiation in normal and abnormal conditions (i.e., cancer). Multivariate flow cytometric analysis, indexed cell sorting, list mode data processing, clonogenic assays, and statistical analysis of multivariate data will be used to identify probes for clonogenic cells. First, a cocktail of monoclonal antibodies, directed against cell surface antigens, will be developed which when labeled with a single fluorophore, will recognize non-clonogenic immature and differentiated cells. This will be accomplished by a two step screen of antibodies produced by immunizations with immature mouse bone marrow cells. The primary screen is bivariate flow cytometric analysis of antibody cocktail vs. hybridoma supernatant and selection of antibodies that detect unique subpopulations. In the secondary screen the reactivity of antibodies with clonogenic cells is determined. The precise antibody binding characteristics of granulocyte-macrophage progenitors (CFU-GM), high proliferative potential myeloid progenitors (HPP-CFC), and stem cells (S-cells) will be obtained by indexed sorting. If the antibody does not react with clonogenic cells it is added to the existing cocktail. Antibodies positive for clonogenic cells are evaluated by multivariate data analytical techniques for their ability to discriminate between clonogenic compartments. Once discriminants for stem cells and committed progenitors are established, simultaneous flow cytometric analysis of cell surface markers, DNA content and bromodeoxyuridine (BrdUrd) incorporated into DNA will be used to determine the inter-and intra-compartmental kinetic properties of CFU-GM, HPP-CFC, and S-cells. Phase transits and dispersions, growth fraction, compartmental efflux and influx, and inter-compartmental transit times will be determined from bivariate BrdUrd/DNA content distributions of the flow cytometrically-distinct subpopulations at selected intervals following BrdUrd pulse labeling and infusions in vivo. These kinetic data will more clearly define the temporal relationships between cells in each of these clonogenic compartments, and will delineate the types of measurements needed to allow acquisition of similar information in man.
| Neben, S; Redfearn, W J; Parra, M et al. (1991) Short- and long-term repopulation of lethally irradiated mice by bone marrow stem cells enriched on the basis of light scatter and Hoechst 33342 fluorescence. Exp Hematol 19:958-67 |
| Rosette, C D; DeTeresa, P S; Pallavicini, M G (1990) Simultaneous flow cytometric detection of cellular c-myc protein, incorporated bromodeoxyuridine, and DNA. Cytometry 11:547-51 |
| Pallavicini, M G; Haendel, S; Rosette, C et al. (1989) Rapid screening and selection of monoclonal antibodies by bivariate flow cytometric analyses. J Immunol Methods 117:99-106 |
